Intrauterine growth restriction (lUGR) is a significant cause of increased infant mortality and morbidity. A number of placental abnormalities have been described during lUGR including an increase in trophoblast apoptosis. Abnormal trophoblast apoptosis may lead to impaired oxygen and nutrient exchange, suggesting a role for apoptosis in the development of lUGR. To better understand apoptosis in lUGR pregnancies we will utilize an established hyperthermia sheep lUGR model which mimics the human disease considerably. We propose to look at apoptosis in the sheep placentae early in gestation and in sheep exposed to hyperthermia for 20 days, 55 days when placental growth is at its peak and 80 days near term when fetal growth is maximal. Our long-term goals are: 1) to determine the in-vivo placental apoptotic effects, DNA degradation, proliferation rate, telomerase activity and cytokeratin 18 cleavage in our model of hyperthermia induced PI-IUGR in the sheep, 2) to elucidate the expression anti-apoptotic molecules BCL-2 and XIAP and the pro-apoptotic molecules BAX, caspase 3 and caspase 9 in the sheep PI-IUGR placentae. To determine the correlation and expression of HSP27 and of HIF-2a during apoptosis in hyperthermia induced PI-IUGR, and 3) to determine the effect of hyperthermia, hypoxia or both on apoptosis, cytokeratin 18 cleavage, telomerase activity and expression of BCL-2, XIAP, BAX, caspase 3 and 9, HSP27 and HIF-2a in cultured placental binucleated cells (BNC) obtained from normal sheep placentomes. In order to accomplish these we will determine apoptosis using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique (TUNEL) and cytokeratin 18 cleavage expression. Also we will determine the telomerase activity associated with our model of lUGR using the Telomerase Repeat Amplification Protocol (TRAP) technique. To further study apoptosis, we will determine the protein levels of Bcl-2, Bax, and caspase 3 and 9 activation in placentae from animals treated with hyperthermia as compared to controls. Also, we will determine the effects of hyperthermia in the expression of XIAP and HIF-2a in the placentae from treated animals. To study apoptosis in-vitro we will determine the effects of hyperthermia and hypoxia on cultured placental BNCs.